Compound useful for the treatment of nonsense-mutation-mediated diseases and pharmaceutical composition comprising said compound

ABSTRACT

The present invention relates to the compound of formula (I) 
     
       
         
         
             
             
         
       
     
     for use in the treatment of a nonsense-mutation-mediated genetic disease.

The present invention relates to a compound used for treating,preventing or diagnosing a genetic disease caused by a nonsense mutationalso called “nonsense-mutation-mediated disease”. The present inventionalso relates to a pharmaceutical composition comprising theabove-mentioned compound. The present invention further relates to amethod for determining the presence of a nonsense mutation in a givengene.

A nonsense mutation is a genetic in-frame mutation leading to thetransformation of a sense codon into a premature stop codon in themessenger RNA (hereinafter mRNA). A premature stop codon (hereinafterPTC) is defined as a stop codon located in the coding sequence of agene, upstream from the normal stop codon. The normal stop codon stopsthe gene translation and enables a full-length wild type proteinsynthesis. The PTC prevents the wild-type protein synthesis and leads tothe silencing of the mutated gene. The lack of protein (partial or totallack) leads to the pathology. For example, a PTC in the gene coding forthe dystrophin protein causes nonsense-mutation-mediated-DuchenneMuscular Dystrophy in boys.

It is known that nonsense-mutation-mediated prostatic cancers are causedby a PTC in JAK1, SYNJ2 or CLPTM1 genes (see “Identification ofinactivating mutations in the JAK1, SYNJ2 and CLPTM1 genes in prostatecancer cells, using inhibition of nonsense-mediated decay and microarrayanalysis” in Cancer genetics and cytogenetics (2005)). It is also knownthat nonsense-mutation-mediated epilepsy is caused by PTC in GABA_(A)receptor subunit gene (see “Making sense of nonsense GABA_(A) receptormutations associated with genetic epilepsies” in Cell (2010)).

Document WO 2008/101935 discloses compounds useful for treatingnonsense-mutation-mediated diseases. The compounds disclosed in thisdocument are indole derivatives and have been tested on HeLa cancerouscells previously transfected with two plasmids and on two lymphoblasticcell-lines coming from DMD (Duchene muscular dystrophy) patients.Document WO 2004/091502 discloses compounds useful in the treatment ofgenetic diseases caused by a PTC coming from a nonsense mutation. One ofthe compounds disclosed in WO 2004/091502 (ataluren) is currentlystudied in a phase 2a clinical trial as an oral treatment fornonsense-mutation-mediated hemophilia A and B, in a phase 2 trial innonsense-mutation-mediated Methylmalonic Acidemia and in a phase 3 trialin subjects with nonsense-mutation-mediated cystic fibrosis. A phase 2atrial in subjects with nonsense-mutation-mediated Duchenne/Beckermuscular dystrophy has been already completed.

One purpose of the present invention is to provide a compound enablingthe treatment of genetic diseases, caused by a nonsense mutation.

Accordingly, the present invention provides the compound of formula (I)

or a salt, solvate, clathrate, hydrate or polymorph thereof for use inthe treatment or the prophylaxis of a genetic disease, said geneticdisease being a nonsense-mutation-mediated disease.

The compound of Formula (I)((2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylicacid) is commonly called amlexanox. It is currently used as therapeuticagent for the treatment of asthma and allergic rhinitis whenadministered orally, and for the treatment of canker sores when usedtopically.

It is also used for the treatment of aphthous ulcer (Aphthasol®). It isknown for its anti-inflammatory and anti-allergic properties. It is alsoused in the treatment of bronchic asthma. Its safety profile is wellknown.

WO2009/151569 relates to a method of treating a B-cell proliferativedisorder by administering to a patient a BAR agonist (Beta(2)-adrenergicreceptor). The BAR agonist may be administered as a monotherapy or incombination with one or more other agents, like a PDE enzyme inhibitor.The compound according to the invention is cited in a long list ofcompounds as a PDE inhibitor. WO2009/151569 does not indicate or suggestthat a genetic mutation is implicated in the aforementionedproliferative mechanism.

WO 2008/021210 relates to a method for treating a neurodegenerativedisorder using compounds listed in various tables. The compoundaccording to the present invention is disclosed in table la as anantihistamine (H1), a leukotriene receptor antagonist, an antiallergicand an anti-inflammatory. WO 2008/021210 does not disclose nor suggestthat a genetic mutation plays a role in the treated diseases.

Document WO 2010/139985 discloses the use of the compound according tothe present invention for treating a disease associated withneutrophilia. Neutrophilia is a term used for a patient condition whensaid patient has a high number of neutrophil granulocytes in his blood.Neutrophils are the most abundant type of white blood cells in mammals.Neutrophils are associated with inflammatory diseases. Neutrophils arethe first response in the inflammatory process, particularly, inbacterial infection, environmental exposure and some cancers. WO2010/139985 does not disclose nor suggest the use of the above-mentionedcompound for the treatment of a nonsense-mutation-mediated disease.

Moreover, document US 2007/0135473 discloses the use of the compoundaccording to the present invention for treating diseases associated withtumor cells which express one or more proteins of S100 family. Theabove-mentioned compound is considered as effective in retarding theprogression and/or the metastasis of these tumors. S100 proteins havebeen implicated in a variety of intracellular and extracellularfunctions. S100 proteins are involved in regulation of proteinphosphorylation, transcription factors, Ca++ homeostasis, the dynamicsof cytoskeleton constituents, enzyme activities, cell growth anddifferentiation, and the inflammatory response. However, US 2007/0135473does not disclose nor suggest the use of the compound of Formula (I) forthe treatment of a nonsense-mutation-mediated disease. Further, there isno evidence that S100 proteins are involved in a disease caused by anonsense-mutation.

The Applicants have discovered that the compound of Formula (I) is ableto inhibit nonsense-mediated mRNA decay (hereinafter called NMD) and/orhas a readthrough effect, depending on mutated genes, as explainedhereinafter. Accordingly, the compound of the invention enablesfunctional protein synthesis.

Theoretically, when a PTC is present on an mRNA, ribosomes shouldsynthesize a truncated protein. Truncated proteins have a shorterpeptide chain compared to wild-type proteins. A wild-type protein isdefined as a protein synthesized when the corresponding gene does notcomprise any mutations. Truncated proteins may be functional or not. Inother words, truncated proteins may produce or not the same effects oncells or entire body as the effects produced by wild-type proteins.mRNAs containing a PTC are eradicated by the organism beforesteady-state translation occurs. This PTC-containing mRNA decay is alsocalled nonsense-mediated mRNA decay (NMD). NMD is a natural qualitativesurveillance mechanism existing in all eukaryote organisms. NMD aims thedegradation of PTC-containing mRNAs.

In some cases, the truncated protein that would come from thetranslation of a nonsense-mutation containing gene would be functional.The truncated-protein functionality may occur despite the fact that thetruncated protein is shorter than the wild type protein. In other words,even if the truncated protein is different from the wild-type one, itmay produce in cells and organism the same effects as those produced bythe wild-type protein. In these cases, the truncated proteins producethe wild-type phenotype. Consequently, in this case, if NMD isinhibited, the functional truncated proteins stay in the cell and havethe same function in the cell and thus organism as the wild-typeprotein, just as if the corresponding gene does not harbor any nonsensemutation.

NMD mechanism is well known and described in several publications (SeeConti, E. and E. Izaurralde, Nonsense-mediated mRNA decay: molecularinsights and mechanistic variations across species. Curr Opin Cell Biol,2005. 17(3): p. 316-25 and Maquat, L. E., Nonsense-mediated mRNA decay:splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol, 2004.5(2): p. 89-99.

It is known that under specific conditions, despite the presence of atermination codon (premature or not) harbored in mRNA, ribosome keeps ontranslating said mRNA into protein by incorporating an amino-acid at thestop codon position. This phenomenon is called “readthrough”. The aminoacid incorporated at the nonsense position can be identical to the aminoacid present in the wild-type protein or different. Therefore, theresulting protein may be functional or not depending on the role of theamino acid located at the nonsense position, for the protein function.

In the last years, there has been an attempt to develop pharmacologicalapproaches for mutations generating in-frame PTCs. These therapeuticapproaches are aimed at promoting translational readthrough of the PTCs,to enable the synthesis and expression of full-length functionalproteins at sufficient levels. In most of these studies, the readthroughdrugs were aminoglycosides, mainly gentamicin. The clinical benefit ofgentamicin is limited since high concentrations and/or long-termtreatments have severe side effects such as kidney damage and loss ofhearing. Recently, PTC124 or ataluren has been identified as a smallorganic compound able to promote readthrough of PTCs. WO2004/091502discloses PTC124 and 1,2,4-oxadiazole benzoic acid derivatives.

According to the present invention, “NMD inhibition” means partial ortotal inhibition of NMD and “PTC readthrough” means partial or totalreadthrough of PTC. It has been found that the compound according to theinvention has a readthrough effect. The compound according to theinvention can therefore be used for the treatment of a genetic diseasecaused by a nonsense-mutation (also called nonsense-mutation-mediateddisease). Said nonsense-mutation is defined as a NMD activatingmutation.

According to the present invention, a “functional protein” is defined asa truncated or not truncated protein able to have the same cellularfunctions as the wild-type protein. When the compound according to theinvention inhibits NMD, a functional truncated protein may be obtained.When the compound according to the invention promotes readthrough of thePTC, a functional not truncated protein may be obtained. NMD inhibitioncan be a complete inhibition or only a partial inhibition. In the caseof a partial inhibition, the compound according to the invention may notcompletely suppress the NMD. When the compound according to theinvention inhibits NMD partially or totally, the quantity ofFTC-containing mRNA increases, so that readthrough promotion by thecompound may be favored.

It is known that a small amount of functional protein may be sufficientto enable a normal activity in cells, tissue and/or whole organism.Sometimes a low concentration of functional protein may be enough toobtain a wild-type phenotype in patients. According to the presentinvention, the term “patient” relates to any kind of living body, moreparticularly an animal like a mammal and more particularly a human.

The compound according to the invention enables to treat and/or preventa nonsense-mutation-mediated disease. The compound of the inventionenables to delete or attenuate one or more of the disease symptoms.

The compound according to the invention may be administered to anypatient as defined hereinabove, said patient being affected by a diseaseas defined hereinabove. Salts of the compound of formula (I) can be usedand are not limited according to the invention. For instance, saltsobtained through reacting compound of formula (I) with an alkalinecompound are also encompassed in the present invention. Sodium,potassium, calcium, magnesium, ammonium salts and chloride may beobtained through such a reaction. Reaction between the compound offormula (I) and at least an organic or inorganic acid may also provideother salts.

According to the present invention, the term polymorph relates to anymixture of amorphous or crystalline forms of the compound according tothe invention.

According to the invention, the disease caused by a nonsense-mutation isnot limited. Many nonsense-mutation-mediated diseases have beendescribed. Many organs or functions of the body may be affected by anonsense-mutation-mediated disease, such as liver or gastro-intestinalfunction, kidney, cardiovascular, pulmonary, muscular, bone marrow,central nervous system functions, metabolism, organogenesis,inflammation and immunity.

For instance, said nonsense-mutation-mediated disease may be chosenamong:

Nonsense-Mutation-Mediated beta-thalassemia, Nonsense-Mutation-MediatedEhlers-Danlos syndrome, Nonsense-Mutation-Mediated Severe myoclonicepilepsy of infancy, Nonsense-Mutation-Mediated achromatopsia,Nonsense-Mutation-Mediated retinitis pigmentosa,Nonsense-Mutation-Mediated Usher Syndrome Type 1C,Nonsense-Mutation-Mediated Adducted thumb-clubfoot syndrome,Nonsense-Mutation-Mediated Alagille syndrome, Nonsense-Mutation-MediatedAlström syndrome, Nonsense-Mutation-Mediated antithrombin deficiency,Nonsense-Mutation-Mediated Carney complex, Nonsense-Mutation-MediatedCurrarino syndrome, Nonsense-Mutation-Mediated Diamond-Blackfan anemia,Nonsense-Mutation-Mediated erythropoietic protoporphyria,Nonsense-Mutation-Mediated Fabry disease, Nonsense-Mutation-Mediatedfactor XIII deficiency, Nonsense-Mutation-Mediated Fanconi-Bickelsyndrome, Nonsense-Mutation-Mediated fish odor syndrome,Nonsense-Mutation-Mediated Gaucher disease, Nonsense-Mutation-MediatedHereditary hemorrhagic telangiectasia, Nonsense-Mutation-Mediatedhomocystinuria, Nonsense-Mutation-Mediated Joubert syndrome and relateddisorders, Nonsense-Mutation-Mediated Krabbe disease,Nonsense-Mutation-Mediated L-2-hydroxyglutaric aciduria,Nonsense-Mutation-Mediated MethylMalonic academia,Nonsense-Mutation-Mediated Niemann-Pick disease,Nonsense-Mutation-Mediated Peters plus syndrome,Nonsense-Mutation-Mediated Townes-Brocks disease,Nonsense-Mutation-Mediated von Willebrand disease,Nonsense-Mutation-Mediated Wiskott-Aldrich syndrome,Nonsense-Mutation-Mediated Kabuki syndrome, Nonsense-Mutation-MediatedChanarin-Dorfman syndrome, Nonsense-Mutation-MediatedLecithin:cholesterol acyltransferase deficiency/fish-eye disease,Nonsense-Mutation-Mediated Marfan Syndrome, Nonsense-Mutation-MediatedMucopolysaccharidiosis, Nonsense-Mutation-mediated Amyloidiosis,Nonsense-Mutation-Mediated Late Infantile Neuronal CeroidLipofuscinosis, Nonsense-Mutation-Mediated coenzyme Q10 Deficiency,Nonsense-Mutation-Mediated Peroxisome biogenesis disorders,Nonsense-Mutation-Mediated lysosomal storage disorders,Nonsense-Mutation-Mediated colorectal cancer, Nonsense-Mutation-Mediatedcongenital enteropeptidase deficiency, Nonsense-Mutation-Mediated CysticFibrosis, Nonsense-Mutation-Mediated Hungarian Peutz-Jeghers Syndrome,Nonsense-Mutation-Mediated Jervell and Lange-Nielsen syndrome,Nonsense-Mutation-Mediated Lynch syndrome, Nonsense-Mutation-Mediatedmicrovillus inclusion disease, Nonsense-Mutation-Mediated Peutz-Jegherssyndrome, Nonsense-Mutation-Mediated xanthinuria,Nonsense-Mutation-Mediated Acidosis, Nonsense-Mutation-Mediated Alportsyndrome, Nonsense-Mutation-Mediated Bardet-Biedl syndrome,Nonsense-Mutation-Mediated Birt-Hogg-Dube syndrome,Nonsense-Mutation-Mediated Dent's disease, Nonsense-Mutation-MediatedGitelman syndrome, Nonsense-Mutation-Mediated Hereditary leiomyomatosisand renal cell cancer, Nonsense-Mutation-Mediated hereditaryspherocytosis, Nonsense-Mutation-Mediated leber congenital amaurosis,Nonsense-Mutation-Mediated Lysinuric protein intolerance,Nonsense-Mutation-Mediated Nephronophthisis, Nonsense-Mutation-Mediatedpolycystic kidney disease, Nonsense-Mutation-Mediatedpseudohypoaldosteronism, Nonsense-Mutation-Mediated renal hypodysplasia,Nonsense-Mutation-Mediated Sporadic clear cell renal cell carcinoma,Nonsense-Mutation-Mediated type 2 papillary renal cell cancers,Nonsense-Mutation-Mediated Urofacial syndrome,Nonsense-Mutation-Mediated von Hippel-Lindau disease,Nonsense-Mutation-Mediated Wilms' tumor, Nonsense-Mutation-MediatedX-linked Alport syndrome, Nonsense-Mutation-Mediated X-linkedhypophosphatemic rickets, Nonsense-Mutation-Mediated Hyperuricaemicnephropathy (juvenile/medullary cystic kidney disease),Nonsense-Mutation-Mediated Tuberous sclerosis,Nonsense-Mutation-Mediated Nephrotic syndrome/congenital nephroticsyndrome, Finnish type Nonsense-Mutation-Mediated Nephrotic syndrome,steroid resistant Nonsense-Mutation-Mediated Nephrotic syndrome 3, earlyonset Nonsense-Mutation-Mediated Nephrotic syndrome/Pierson syndrome,Nonsense-Mutation-Mediated Denys-Drash syndrome,Nonsense-Mutation-Mediated Nephrotic syndrome/Schimke immuno-osseousdysplasia, Nonsense-Mutation-Mediated Primary glucocorticoid resistance,Nonsense-Mutation-Mediated X-linked hypophosphatemia,Nonsense-Mutation-Mediated Primary hyperoxaluria type 1,Nonsense-Mutation-Mediated pseudohypoaldosteronism type 1,Nonsense-Mutation-Mediated proximal renal tubular acidosis,Nonsense-Mutation-Mediated Abetalipoproteinemia and Homozygous FamilialHypobetalipoproteinemia, Nonsense-Mutation-Mediated Alpers syndrome,Nonsense-Mutation-Mediated carbamyl phosphate synthetase I deficiency,Nonsense-Mutation-Mediated Cholesteryl Ester Storage Disease,Nonsense-Mutation-Mediated citrin deficiency, Nonsense-Mutation-MediatedDubin-Johnson syndrome, Nonsense-Mutation-Mediated erythropoieticprotoporphyria, Nonsense-Mutation-Mediated Factor V deficiency,Nonsense-Mutation-Mediated Glycogen storage disease,Nonsense-Mutation-Mediated Hemophilia A (factor VIII Deficiency),Nonsense-Mutation-Mediated Hemophilia B (factor IX Deficiency),Nonsense-Mutation-Mediated hepatocellular carcinoma,Nonsense-Mutation-Mediated Hepatoerythropoietic porphyria,Nonsense-Mutation-Mediated hereditary spastic paraplegias,Nonsense-Mutation-Mediated Hypobetalipoproteinemia,Nonsense-Mutation-Mediated Inherited factor XI deficiency,Nonsense-Mutation-Mediated Maturity-onset diabetes of the young,Nonsense-Mutation-Mediated microcytic anemia and iron deficiency,Nonsense-Mutation-Mediated mitochondrial DNA depletion,Nonsense-Mutation-Mediated mitochondrial DNA depletion syndrome,Nonsense-Mutation-Mediated phenylketonuria, Nonsense-Mutation-Mediatedpolycystic liver disease, Nonsense-Mutation-Mediated porphyria cutaneatarda, Nonsense-Mutation-Mediated progressive familial intrahepaticcholestasis, Nonsense-Mutation-Mediated Wilson Disease,Nonsense-Mutation-Mediated autosomal dominant hypercholesterolemia,Nonsense-Mutation-Mediated factor XII Deficiency,Nonsense-Mutation-Mediated factor X Deficiency,Nonsense-Mutation-Mediated hypofibrinogenaemia,Nonsense-Mutation-Mediated Afibrinogenaemia, Nonsense-Mutation-Mediatedfactor VII deficiency, Nonsense-Mutation-Mediated agammaglobulinemia,Nonsense-Mutation-Mediated amegakaryocytic thrombocytopenia,Nonsense-Mutation-Mediated dyserythropoietic anemia type II,Nonsense-Mutation-Mediated Duchenne and Becker Muscular Dystrophy,Nonsense-Mutation-Mediated Centronuclear myopathies,Nonsense-Mutation-Mediated limb girdle muscular dystrophy or Miyoshimyopathy, Nonsense-Mutation-Mediated Ullrich disease,Nonsense-Mutation-mediated Spinal muscular atrophy,Nonsense-Mutation-Mediated dystrophic epidermolysis bullosa,Nonsense-Mutation-Mediated Hailey-Hailey Disease,Nonsense-Mutation-Mediated Herlitz junctional epidermolysis bullosa, andNonsense-Mutation-Mediated Netherton syndrome.

In one embodiment, the nonsense-mutation-mediated disease is selectedfrom beta-thalassemia, Marfan synfrome, Duchenne Muscular Dystrophy,Becker Muscular Dystrophy, Ullrich disease, Hurler syndrome, cancer,cystic fibrosis, Spinal muscular atrophy, amylosis, LINCL (LateInfantile Neuronal Ceroid Lipofuscinosis), Haemophilia, Alzheimer'sdisease, Atherosclerosis, Gigantism, Dwarfism, Hypothyroidism,Hyperthyroidism, Obesity, Parkinson's disease, Niemann Pick disease,Family hypercholesterolemia, and retinitis pigmentosa.

Several studies have shown that the compound of Formula (I) ispreferably distributed in liver, kidneys and intestine. The lungs, theheart, the muscles and the skin are slightly less exposed by thecompound of Formula (I) after oral administration. Considering thephysiopathology of the diseases and the affected organs for each diseasecited hereinabove, and according to the present invention, the saidnonsense-mutation-mediated diseases may be preferably chosen among:

Diseases Affecting the Liver:

Nonsense-Mutation-Mediated Abetalipoproteinemia and Homozygous FamilialHypobetalipoproteinemia, Nonsense-Mutation-Mediated Alpers syndrome,Nonsense-Mutation-Mediated carbamyl phosphate synthetase I deficiency,Nonsense-Mutation-Mediated Cholesteryl Ester Storage Disease,Nonsense-Mutation-Mediated citrin deficiency, Nonsense-Mutation-MediatedDubin-Johnson syndrome, Nonsense-Mutation-Mediated erythropoieticprotoporphyria, Nonsense-Mutation-Mediated Factor V deficiency,Nonsense-Mutation-Mediated Glycogen storage disease,Nonsense-Mutation-Mediated Hemophilia A (factor VIII Deficiency),Nonsense-Mutation-Mediated Hemophilia B (factor IX Deficiency),Nonsense-Mutation-Mediated hepatocellular carcinoma,Nonsense-Mutation-Mediated Hepatoerythropoietic porphyria,Nonsense-Mutation-Mediated hereditary spastic paraplegias,Nonsense-Mutation-Mediated Hypobetalipoproteinemia,Nonsense-Mutation-Mediated Inherited factor XI deficiency,Nonsense-Mutation-Mediated Maturity-onset diabetes of the young,Nonsense-Mutation-Mediated microcytic anemia and iron deficiency,Nonsense-Mutation-Mediated mitochondrial DNA depletion,Nonsense-Mutation-Mediated mitochondrial DNA depletion syndrome,Nonsense-Mutation-Mediated phenylketonuria, Nonsense-Mutation-Mediatedpolycystic liver disease, Nonsense-Mutation-Mediated porphyria cutaneatarda, Nonsense-Mutation-Mediated progressive familial intrahepaticcholestasis, Nonsense-Mutation-Mediated Wilson Disease,Nonsense-Mutation-Mediated autosomal dominant hypercholesterolemia,Nonsense-Mutation-Mediated factor XII Deficiency,Nonsense-Mutation-Mediated factor X Deficiency,Nonsense-Mutation-Mediated hypofibrinogenaemia,Nonsense-Mutation-Mediated Afibrinogenaemia, Nonsense-Mutation-Mediatedfactor VII deficiency,

Diseases Affecting the Kidneys:

Nonsense-Mutation-Mediated Acidosis, Nonsense-Mutation-Mediated Alportsyndrome, Nonsense-Mutation-Mediated Bardet-Biedl syndrome,Nonsense-Mutation-Mediated Birt-Hogg-Dube syndrome,Nonsense-Mutation-Mediated Dent's disease, Nonsense-Mutation-MediatedGitelman syndrome, Nonsense-Mutation-Mediated Hereditary leiomyomatosisand renal cell cancer, Nonsense-Mutation-Mediated hereditaryspherocytosis, Nonsense-Mutation-Mediated leber congenital amaurosis,Nonsense-Mutation-Mediated Lysinuric protein intolerance,Nonsense-Mutation-Mediated Nephronophthisis, Nonsense-Mutation-Mediatedpolycystic kidney disease, Nonsense-Mutation-Mediatedpseudohypoaldosteronism, Nonsense-Mutation-Mediated renal hypodysplasia,Nonsense-Mutation-Mediated Sporadic clear cell renal cell carcinoma,Nonsense-Mutation-Mediated type 2 papillary renal cell cancers,Nonsense-Mutation-Mediated Urofacial syndrome,Nonsense-Mutation-Mediated von Hippel-Lindau disease,Nonsense-Mutation-Mediated Wilms' tumor, Nonsense-Mutation-MediatedX-linked Alport syndrome, Nonsense-Mutation-Mediated X-linkedhypophosphatemic rickets, Nonsense-Mutation-Mediated Hyperuricaemicnephropathy (juvenile/medullary cystic kidney disease),Nonsense-Mutation-Mediated Tuberous sclerosis,Nonsense-Mutation-Mediated Nephrotic syndrome/congenital nephroticsyndrome, Finnish type, Nonsense-Mutation-Mediated Nephrotic syndrome,steroid resistant, Nonsense-Mutation-Mediated Nephrotic syndrome 3,early onset, Nonsense-Mutation-Mediated Nephrotic syndrome/Piersonsyndrome, Nonsense-Mutation-Mediated Denys-Drash syndrome,Nonsense-Mutation-Mediated Nephrotic syndrome/Schimke immuno-osseousdysplasia, Nonsense-Mutation-Mediated Primary glucocorticoid resistance,Nonsense-Mutation-Mediated X-linked hypophosphatemia,Nonsense-Mutation-Mediated Primary hyperoxaluria type 1,Nonsense-Mutation-Mediated pseudohypoaldosteronism type 1,Nonsense-Mutation-Mediated proximal renal tubular acidosis.

Diseases Affecting the Intestine:

Nonsense-Mutation-Mediated colorectal cancer, Nonsense-Mutation-Mediatedcongenital enteropeptidase deficiency, Nonsense-Mutation-Mediated CysticFibrosis, Nonsense-Mutation-Mediated Hungarian Peutz-Jeghers Syndrome,Nonsense-Mutation-Mediated Jervell and Lange-Nielsen syndrome,Nonsense-Mutation-Mediated Lynch syndrome, Nonsense-Mutation-Mediatedmicrovillus inclusion disease, Nonsense-Mutation-Mediated Peutz-Jegherssyndrome, Nonsense-Mutation-Mediated xanthinuria.

Diseases Affecting Several Organs:

Nonsense-Mutation-Mediated beta-thalassemia, Nonsense-Mutation-MediatedEhlers-Danlos syndrome, Nonsense-Mutation-Mediated Adductedthumb-clubfoot syndrome, Nonsense-Mutation-Mediated Alagille syndrome,Nonsense-Mutation-Mediated Alström syndrome, Nonsense-Mutation-Mediatedantithrombin deficiency, Nonsense-Mutation-Mediated Carney complex,Nonsense-Mutation-Mediated Currarino syndrome,Nonsense-Mutation-Mediated Diamond-Blackfan anemia,Nonsense-Mutation-Mediated erythropoietic protoporphyria,Nonsense-Mutation-Mediated fabry disease, Nonsense-Mutation-Mediatedfactor XIII deficiency, Nonsense-Mutation-Mediated Fanconi-Bickelsyndrome, Nonsense-Mutation-Mediated fish odor syndrome,Nonsense-Mutation-Mediated Gaucher disease, Nonsense-Mutation-MediatedHereditary hemorrhagic telangiectasia, Nonsense-Mutation-Mediatedhomocystinuria, Nonsense-Mutation-Mediated Joubert syndrome and relateddisorders, Nonsense-Mutation-Mediated Krabbe disease,Nonsense-Mutation-Mediated L-2-hydroxyglutaric aciduria,Nonsense-Mutation-Mediated MethylMalonic acidemia,Nonsense-Mutation-Mediated Niemann-Pick disease,Nonsense-Mutation-Mediated Peters plus syndrome,Nonsense-Mutation-Mediated Townes-Brocks disease,Nonsense-Mutation-Mediated von Willebrand disease,Nonsense-Mutation-Mediated Wiskott-Aldrich syndrome,Nonsense-Mutation-Mediated Kabuki syndrome, Nonsense-Mutation-MediatedChanarin-Dorfman syndrome, Nonsense-Mutation-MediatedLecithin:cholesterol acyltransferase deficiency/fish-eye disease,Nonsense-Mutation-Mediated Marfan Syndrome, Nonsense-Mutation-MediatedMucopolysaccharidiosis, Nonsense-Mutation-mediated Amyloidiosis,Nonsense-Mutation-Mediated Late Infantile Neuronal CeroidLipofuscinosis, Nonsense-Mutation-Mediated coenzyme Q10 Deficiency,Nonsense-Mutation-Mediated Peroxisome biogenesis disorders,Nonsense-Mutation-Mediated lysosomal storage disorders.

Considering that the compound according to Formula (I) can beadministered topically, and according to the present invention, the saidnonsense-mutation-mediated diseases may be also chosen among thefollowing diseases affecting the skin or the eyes:

-   Nonsense-Mutation-Mediated achromatopsia, Nonsense-Mutation-Mediated    retinitis pigmentosa, Nonsense-Mutation-Mediated Usher Syndrome Type    1C, Nonsense-Mutation-Mediated dystrophic epidermolysis bullosa,    Nonsense-Mutation-Mediated Hailey-Hailey Disease,    Nonsense-Mutation-Mediated Herlitz junctional epidermolysis bullosa,    Nonsense-Mutation-Mediated Netherton syndrome.

Usually, the compound according to the present invention, either alone(in any of the forms described above) or in combination with anotheractive agent, is administrated to patients in admixture with one or morepharmaceutically acceptable excipient(s). The excipients are suitablychosen depending on the mode of administration and the excipientinfluence on the compound's solubility and stability. The galenic formmay be also relevant.

Consequently, a second aspect of the invention relates to apharmaceutical composition for use in the treatment and/or prophylaxisof a genetic disease, said genetic disease being anonsense-mutation-mediated disease. The pharmaceutical compositionaccording to the invention comprises, as an active ingredient, at leastthe compound of formula (I) and one or more pharmaceutically acceptableexcipient(s). The composition according to the present inventioncontains the compound of the invention and thus has the sametoxicological properties as the compound itself. It is furthermore notedthat the toxicity of the compound of the invention has already beenevaluated for an oral administration; the toxicity of an injectableformulation should not differ since what is assessed is the toxicity ofthe molecule itself optionally metabolised in blood. Accordingly, thecomposition of the invention can be quickly used and commercialised as adrug.

According to one embodiment, the composition according to the inventioncan be injected. In this case, the excipient is liquid. The activemolecule is dissolved or suspended in the excipient. However, thecomposition according to the invention is not limited to this type ofcomposition.

The present invention also relates to a pharmaceutical composition asdescribed herein above, further comprising a readthrough agent. Thereadthrough agent is able to promote the PTC readthrough. The PTC isharbored in the mRNA coming from the transcription of the gene harboringsaid nonsense mutation. The inventors have observed a synergic effectbetween the compound of the invention and at least one particular PTCreadthrough molecule.

Advantageously, the readthrough agent is3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl] benzoic acid (PTC124disclosed in WO 2004/091502). This readthrough agent is not veryefficient and strong doses have to be administered to patients (16 to 40mg/kg/day). The synergism between this readthrough agent and thecompound according to the invention may enhance the activity of saidreadthrough agent in some cases. Accordingly, lower doses of saidreadthrough agent are needed.

Accordingly, another aspect of the invention relates to a combination of(i) a compound of formula (I):

or a salt, solvate, clathrate, hydrate or polymorph thereof, and (ii)3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl] benzoic acid. The activepharmaceutically agents (i) and (ii) can be administered simultaneously,sequentially or over a period of time.

However, according to the invention, any other readthrough agent mayalso be mixed or combined with the compound according to the presentinvention or with the combination PTC124/compound of Formula (I). Thisreadthrough agent is thus included in the composition according to theinvention. The aforementioned readthrough agent may also be separatelyconditioned in order to be administrated with the molecule of theinvention, at the same time or according to a delayed administration.

The Man skilled in the Art is able to determine whether a disease iscaused by a nonsense-mutation. It is possible, for a given protein, tostudy whether said protein concentration is relevant towards a disease.The Man skilled in the Art is able to determine whether a given diseasecaused by a too low protein concentration is anonsense-mutation-mediated disease by following the hereinaftermentioned steps:

Identification of the gene coding for said given protein andidentification of the mRNA coming from said gene transcription; and

Comparison of normal genes or normal mRNAs with genes and mRNAs comingfrom a patient suffering from said disease.

The present invention also relates to a method for determining whetherthere is a nonsense-mutation harbored in a given gene. This given geneis transcribed into mRNA. The transcribed mRNA harbors a FTC and codesfor a given known protein. According to the method of the presentinvention,

Patient's cells are incubated with the compound or a composition of theinvention in order to obtain RNA synthesis;

Synthesized RNAs and/or proteins are extracted from the cells andpurified;

Purified RNAs are then reverse-transcribed and amplified in order toenable RNA quantitation.

In one embodiment the patient's cells are cells from an organ thought tobe affected by the nonsense-mutation or cells differentiated frominduced pluripotent stem cells issued from the patient. In anotherembodiment an increased quantity of purified RNAs and/or proteinsindicates NMD inhibition and/or readthrough and therefore the presenceof a nonsense-mutation.

The method according to the invention therefore enables to determinewhether the genetic disease is at least partially caused by a nonsensemutation. Accordingly, this enables treatment adjustment.

In another aspect, the invention relates to a method of assessingwhether the compound of formula (I) or a pharmaceutical compositioncontaining it is efficient in treating a patient diagnosed with anonsense-mutation-mediated genetic disease, said method comprising thesteps of:

treating the patient with said compound or said composition;

observing at least one disease marker in the patient, or extractingsynthesized RNAs and/or proteins from said patient cells wherein amodification of the disease marker(s) or an increased quantity ofpurified RNAs and/or proteins indicates NMD inhibition and/orreadthrough, hence that the treatment is efficient.

A disease marker may be the protein (truncated or not) which is normallynot synthesized because of the PTC. A disease marker may also be anyother substance involved in the disease mechanism and particularly anysubstance usually used for the diagnosis of the disease. For instance,in the case of hemophilia A, a marker may be Factor VIII, in the case ofhemophilia B, a marker may be factor IX, in the case of Duchene musculardystrophy, a marker may be dystrophin itself, in the case of cysticfibrosis, a marker may be the concentration of chloride that is excretedin sweat.

The present invention also relates to the use of the compound of Formula(I) in the manufacture of a drug for treating and/or preventingnonsense-mutation-mediated genetic diseases.

The present invention also relates to a therapeutic method for treatingnonsense-mutation-mediated genetic disease comprising the step ofadministering a compound according to formula (I) and/or apharmaceutical composition according to the invention.

The present invention, its features and advantages will be explained onthe basis of the following Examples and Figures, wherein:

FIG. 1 shows the NMD inhibiting activity of a composition according tothe present invention in HeLa cells, using Firefly luciferase;

FIG. 2 shows P53 mRNA stabilisation in Calu-6 cells (cells coming from apatient suffering from a genetic nonsense-mediated lung cancer) vs.active molecule concentration, said stabilisation being obtained throughNMD inhibition by a composition according to the invention;

FIG. 3 is a graph showing the ratio P53 mRNA/GADPH mRNA vs. the activemolecule concentration of a composition according to the invention;

FIG. 4 shows the full-length P53 protein synthesized in Calu-6 cells inthe presence of a composition according to the invention;

FIG. 5 shows mRNA MOR concentration in mouse brain cells vs. activecompound concentration in a mouse model wherein MOR gene harbors anonsense-mutation;

FIG. 6 shows the synergism between the composition of the invention andPTC124 (premature stop codon readthrough agent);

FIG. 7 shows CFTR mRNA in IB3 cells (cells coming from a patientsuffering from nonsense-mutation mediated cystic fibrosis) incubatedwith compositions according to the present invention having differentactive molecule concentrations;

FIG. 8 shows CFTR mRNA concentration in IB3 cells incubated in thepresence of a composition according to the present invention vs. theactive molecule concentration of said composition;

FIG. 9 shows P53 mRNA in Calu-6 cells incubated with three differentcompositions, each composition containing 25 μmol/l of active compoundaccording to the invention;

FIG. 10 shows the dose-response effect of the compound of the inventionon iodide efflux in 6-CFSMEo⁻ cells (cells coming from a patientsuffering from nonsense-mutation mediated cystic fibrosis); and

FIGS. 11a and b show the effect of several doses of the compound of theinvention on the expression of PTC-containing dystrophin gene in DMDcells coming from a patient suffering from nonsense-mediated Duchennemuscular dystrophy.

EXAMPLES

In the following examples the compound of formula (I) will be referredto as molecule G07. Related compounds G08 and G09 were also used intests.

Example 1 Determination of NMD Inhibition by Using Firefly Luciferase

The presence of one of the following UPF proteins (UPF1, UPF2, UPF3(also called UPF3a) or UPF3X (also called UPF3b)) on the 3′UTR part ofan mRNA, activates NMD on this particular mRNA. When an UPF protein islocated downstream from a normal stop codon, said normal stop codon isrecognized as a PTC. Consequently, the mRNA comprising the UPF proteinwill be degraded through NMD.

HeLa cell-line expressing Firefly luciferase mRNA were used. Fireflyluciferase mRNA harbors MS2 binding sites in the 3′UTR. Theabove-mentioned cells further contain a fusion protein which consists insaid MS2 protein and one of the four UPF proteins. This fusion proteinenables UPF positioning downstream from the normal stop codon (i.e. onthe 3′UTR part of this mRNA). Accordingly, the normal stop codon isrecognized as a PTC. Consequently, NMD is activated and degrades Fireflyluciferase mRNA.

Luciferase activity in the above-mentioned cells is thus directly linkedto NMD activity. A low luminescence indicates that NMD is activated anda high luminescence indicates NMD inhibition. In the presence of G07,luciferase activity is higher than in control wells. This shows the NMDinhibiting activity of G07.

HeLa cells were cultured in DMEM (Gibco) supplemented with 10% FBS andcontaining 10 μmol/l of G07.

Since G07 inhibits NMD regardless the type of UPF protein located onmRNA, G07 can be considered as a general NMD inhibitor.

Example 2 Beneficial Effect of G07 on Cells from Patient HarboringNonsense-Mutation-Mediated Lung Cancer

Calu-6 cells (ATCC) harbor a UGA PTC at the codon 196 of the P53 gene.These Calu-6 cells have been isolated from a lung-cancer patient. RNAsof said Calu-6 cells were reverse transcribed and amplified through PCR(RT-PCR) in the presence of a radioactive labeling nucleotide in orderto measure P53 mRNA and GAPDH mRNA levels (GAPDH being used as a loadingcontrol). Calu-6 cells harbor a nonsense mutation in the P53 gene.Accordingly, the P53 gene is not expressed in Calu-6 cells because ofthe NMD activity on P53 mRNA. Amplified reverse transcribed mRNAs wereloaded on an acrylamide gel and submitted to electrophoresis. Acrylamidegel was then dried on Whatman paper and applied on a phosphoscreen(Kodak). A Personal Molecular Imager (Bic-Rad) was used to quantifyamplified radiolabelled species. Calu-6 cells were incubated withgrowing concentrations of G07 in DMSO during about twenty hours. Thiswas carried out in order to confirm NMD inhibition activity of G07.After incubation, RNAs were purified by using TriReagent (MRC) and thenreverse-transcribed into cDNA which can be PCR amplified in presence ofa radiolabelled nucleotide. Corresponding results are shown in FIG. 2.

FIG. 2 shows an increased quantity of P53 mRNA when the concentration ofG07 increases. P53 mRNA is normally degraded via NMD. Thus, anincreasing quantity of P53 mRNA results from stabilization thereof.Accordingly, G07 seems to have a dose response activity on NMDmechanism. This experiment confirms NMD inhibiting activity of G07 andfurther proves that. G07 is able to inhibit NMD towards an endogenousmRNA. GAPDH (Glyceraldehyde 3-phosphate deshydrogenase) mRNA wasamplified in order to normalize the measurement of P53 mRNA between twolanes.

FIG. 3 shows that the higher the G07 concentration, the greater theamount of P53 mRNA in cells.

Moreover, G07 enables the synthesis of a truncated P53 protein and afull-length P53 protein in Calu-6 cells. G07 is thus different from themolecules disclosed in WO 2008/101935. Regarding the molecules disclosedin the above-mentioned document, only the truncated protein has beendetected. This result is shown in FIG. 4. Protein extracts of Calu-6cells incubated or not with G07 were loaded on an electrophoresis geland then transferred on a nitrocellulose membrane in order to detect P53protein after incubation with an anti-P53 antibody (an anti-P53 antibodyis an antibody directed against the amino terminal part of P53 (SantaCruz)). The obtained result is shown in FIG. 4. According to FIG. 4,when Calu-6 cells are incubated with G07 during about 20 hours,full-length and truncated P53 proteins are synthesized by cells.

FIG. 4 also shows an analysis of the presence of P21 protein in order tocheck whether the full-length P53 protein is functional. When P53protein is synthesized, P53 activates P21 expression. Western-Blotanalysis shows the presence of P21 when increasing G07 concentrationsare used (25 and 125 μM). Thus, P53 protein synthesized thanks to G07induces P21 expression as WT P53 protein does. Consequently, theApplicants have thus confirmed that G07 enables the synthesis of afunctional full-length P53 protein.

Consequently, G07 enables full-length protein synthesis. G07 may enablePTC readthrough. G07 may also inhibit NMD at a critical stage thereofenabling therefore the full-length protein synthesis. The synthesizedfull-length protein may be the wild-type protein.

Example 3 In Vivo NMD Inhibition

In KIM mice, MOR gene harbors a NMD-activating nonsense-mutation. MORgene is expressed in nervous central system. Solutions containing 0.2, 2or 20 mg/kg of G07 (1Q, 2Q or 3Q respectively) dissolved in DMSO wereinjected into KIM mice. A control group consists of animals treated withpure DMSO. Animals were then euthanized 6, 24 or 32 hours afterinjection. RNAs extracted from brain homogenates were purified usingTriReagent (MRC) and then analyzed as described with reference to FIG.2. The difference between the experiments described with reference toFIG. 2 is that in the present case the mRNAs analyzed are the mRNAcoding for MOR gene and the mRNA coding for the GAPDH gene used as acontrol to quantify measured MOR mRNA. The first 3 wells from the leftcorrespond to a half dilution of a sample coming from a wild-type mousebrain. These 3 wells were used to check the PCR conditions which have tobe quantitatively useful.

FIG. 5 shows measurements of mRNA levels of MOR and GADPH (the latterused as a control). The same technique as described with reference toFIG. 2 was used except the fact that RNA comes from KIM mice brains.Some of these KIM mice were injected with G07.

A stabilization of MOR mRNA was observed after 24 hours for 1Q and 2Qconcentrations. This indicates NMD inhibition. Hence, this experimentconfirms the in vivo NMD inhibition activity of G07.

Example 4 Use of G07 in Combination with a Readthrough Agent

PTC124 (ataluren or 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoicacid) is a molecule which activates readthrough. This molecule has noNMD inhibiting activity. Calu-6 cells were incubated in DMSO as acontrol (−), in DMSO solutions containing increasing concentrations ofPTC124 and in DMSO solutions containing increasing concentrations ofPTC124 and further 25 μM of G07. Protein extracts of these cells wereprepared 24 hours after the start of the incubation and analyzed viaWestern Blot. According to the Western-blot as shown in FIG. 6 (i.e.after a very short exposition time) full-length P53 protein can beobserved only in cells incubated with a medium containing G07 andPTC124. Thus, the two molecules have synergism when used together. FIG.6 shows that incubating Calu-6 cells in a medium containing a mixture ofPTC124 and G07 improves full-length P53 protein synthesis. Thisimprovement is more efficient than the improvement obtained for eachmolecule separately used. Thus there is a synergism between G07 andPTC124.

Example 5 NMD Inhibition in IB3 (Cystic Fibrosis Model)

IB3 cells harbor a FTC in position 1282 in place of the tryptophan codonof the CFTR gene.

IB3 cells (ATCC) were incubated in DMSO solutions containing pure DMSO(as a control) or increasing G07 concentrations, respectively, during 20hours.

RNAs obtained after incubation were purified and a RT-PCR was performedto measure the amount of CFTR mRNA (containing a PTC) and the amount ofGADPH mRNA (used as a control). On the basis of the ratio amount of CFTRmRNA/amount of GAPDH mRNA shown, we can conclude that there is anincreased stabilization of CTFR mRNA. This result is shown in FIG. 7.This result confirms the NMD inhibiting activity of G07 as alreadyobserved in Calu-6 cells. This experiment shows the ability of G07 toinhibit NMD in several and different types of cell.

As shown in FIG. 8, the greater the concentration of G07 in the culturemedium, the more stabilized the mRNA normally degraded through NMD.

This indicates that. NMD inhibition depends on the concentration of G07in the culture medium.

Example 6 NMD Inhibition Activity of Compositions Containing G07 G08 andG09, Respectively

Calu-6 cells were incubated with compositions containing 25 μM of G07,G08 and G09, respectively. The same experiment as previously describedin reference with NMD inhibition confirmation in Calu-6 cells wasperformed with each composition. Results are shown in FIG. 9. Each 25 μMcomposition inhibits NMD and enables a stabilization of P53 mRNA inCalu-6 cells.

Example 7 Dose-Response Effect of G07 on Iodide Efflux in 6-CFSMEo⁻Cells (Cells Coming from a Patient Suffering from Cystic Fibrosis)

6-CFSMEo− cells harbor a PTC in place of the glutamine 2 codon of theCFTR gene.

6-CFSMEo⁻ cells were seeded in 96-well plates and loaded overnight with10 mM of the halide-sensitive fluorophore dye6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ, Invitrogen). Cells werewashed twice with iodide buffer (135 mM NaI, 2.4 mM K₂HPO₄, 0.6 mMKH₂PO₄, 1 mM MgSO₄, 1 mM CaSO₄, 10 mM dextrose and 10 mM Hepes, pH 7.3)and then incubated in iodide buffer for 30 min. After basal fluorescencewas measured for 2 min, the iodide buffer was replaced with nitratebuffer (with 135 mM NaNO₃ instead of NaI) containing 20 μM of forskolinand 200 μM of IBMX, and fluorescence was further measured for 10minutes. The fluorescence intensities were measured every 15 secondsusing the Tristar LB 941 microplate reader (Berthold) equipped with a340 nm excitation filter and a 450 nm emission filter. The arrow in FIG.10 shows the addition of nitrate buffer containing 20 μM of forskolinand 200 μM of BMX, as explained above. G07 concentrations used were 0.2μM, 1 μM, 5 μM and 25 μM, respectively. The increase in iodide efflux isshown as mean±SEM from at least four independent assays. It refers tothe fluorescence at the reading time; F0 refers to the averagefluorescence before addition of the above-mentioned nitrate buffercontaining 20 μM of forskolin and 200 μM of IBMX.

On the basis of the results shown in FIG. 10, it is dear that G07 has adose-response effect on 6-CFSMEo− cells and leads to the synthesis offunctional CFTR protein from a PTC-containing CFTR gene.

Example 8 Effect of G07 on the Expression of PTC-Containing DystrophinGene in Cells from a Patient Suffering from Nonsense-Mutation-MediatedDuchenne Muscular Dystrophy (i.e. DMD Cells)

As regards the results of FIG. 11 a, DMD cells were incubated withincreasing amounts of G07 molecule or as a control with DMSO. RNAs werepurified using RNazol reagent (MRC) and reverse transcribed usingSuperscript. II (Life technologies) and random hexamer. PCR usingradiolabelled CTP was performed in order to amplify either dystrophin orGAPDH cDNA. Amplification products were loaded on 5% polyacrylamide gel.The gel was then dried and exposed on phosphoscreen in order to allowquantification of each amplification species by Personal MolecularImager (Biorad).

As regards the results of FIG. 11 b, DMD cells were incubated withincreasing amounts of G07 molecule or as a control with DMSO. Proteinextracts from these cells were loaded on 10% SDS-PAGE and transferred onnitrocellulose membrane. Nitrocellulose membrane was then exposed toprimary antibody (either anti-dystrophin antibody raised against theamino-part of dystrophin protein (Santa-Cruz), or anti-tubulin antibodyin order to specify the loading protein amount of each lane.

FIGS. 11a and b show that G07 is able to rescue the expression ofPTC-containing dystrophin gene in cells coming from a patient sufferingfrom nonsense-mediated Duchenne muscular dystrophy.

As shown in FIG. 11 a, increasing amounts of G07 lead to thestabilization of dystrophin mRNA. This result is obtained throughRT-PCR. The maximum of efficacy is obtained at a concentration of 5 μM.

As shown in FIG. 11 b, increasing amounts of G07 lead to the synthesisof dystrophin protein (results obtained through Western-blot with anantibody raised against the amino-terminal part of dystrophin protein).The maximum of G07 efficacy is 5 μM which is consistent with the resultsshown in FIG. 11 a.

1-21. (canceled)
 22. A method of treating a nonsense-mutation-mediatedlysosomal storage disorder, which comprises administering as sole activeingredient to a subject in need thereof a compound of formula (I):

or a salt, solvate, clathrate, hydrate or polymorph thereof.
 22. Themethod of claim 21, wherein the compound of formula (I) is administeredthrough injection.
 23. A method of treating a nonsense-mutation-mediatedlysosomal storage disorder, which comprises administering to a subjectin need thereof a compound of formula (I):

or a salt, solvate, clathrate, hydrate or polymorph thereof, incombination with an agent able to enhance readthrough of the mRNApremature stop-codon, said mRNA premature stop-codon coming from thetranslation of the gene comprising the nonsense-mutation.
 24. The methodof claim 23, wherein said agent is3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl] benzoic acid.
 25. The methodof claim 23, wherein the compound of formula (I) and the agent areadministered simultaneously.
 26. The method of claim 23, wherein thecompound of formula (I) and the agent are administered sequentially. 27.The method of claim 26, wherein the compound of formula (I) and theagent are administered over a period of time.